Experiments outlined in this proposal will address the roles of protein-protein and RNA-protein interactions in picornavirus infections of human cells.A molecular genetic and biochemical analysis is proposed to characterize in detail the enzyme-substrate interactions between poliovirus 3CD proteinase and its cognate precursor polypeptides. The proposed experimental approaches will focus on 3CD interactions with the P1 capsid precursor polypeptide and will emphasize the importance of genetic as well as structural determinants in effecting the proteolytic cleavage events that produce functional poliovirus gene products. In addition to the production of the viral structural proteins, the 3CD (3C) proteinase activity is required for the production of non-structural proteins that are primarily involved in viral RNA replication. The end products of 3C processing of proteins involved in viral RNA synthesis are assembled into membrane-bound replication complexes in the infected cell. The scope of the present proposal will be expanded (beyond the previous experimental objectives) to include an analysis of the genetic and protein determinants of poliovirus RNA replication. This work will take advantage of our previous studies employing the genetic manipulation of the hydrophobic domain of poliovirus polypeptide 3AB and the rapidly-emerging technologies for analysis of sequence-specific RNA- protein interactions.The results from the proposed experimental approaches are expected to have a direct impact on the elucidation of the genetic determinants of picornavirus gene expression. It is anticipated that these studies will contribute to an understanding of how sequence-specific macromolecular interactions (either at the protein- protein or RNA-protein level) play a role in cellular functions such as prohormone processing, chaperon-aided protein folding, and mRNA splicing.